Toxicity of the vitrification solutions was evaluated by assessing ovarian follicle membrane integrity with

trypan blue staining and the effect of vitrification protocol on the follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 fluorescent probe and confocal microscopy. Zebrafish were maintained in aerated and temperature-regulated (27 °C) water in 40 L aquaria under a light/dark photoperiod of 12/12 h. Fish were fed twice a day with TetraMin® dry flake food (Tetra, Germany) and live brine shrimp (Artemia franciscana) nauplii. To obtain ovarian follicles, BMS 907351 female zebrafish with fully grown ovaries were anesthetized with a lethal dose of tricaine (0.6 mg/ml) followed by decapitation. Ovaries were immediately removed after decapitation and were gently placed into a Petri dish containing 90% Leibovitz L-15 medium (pH 9.0) supplemented with l-glutamine (Sigma). Ovarian tissue fragments containing stage III ovarian follicles were obtained manually by using forceps and fine needles under a dissecting microscope. In this study, follicles of 0.50–0.69 mm in diameter, having an intrafollicular oocyte with a dark ooplasm and a well-marked cell outline (immature

oocytes at late stage III) were used, based on the criteria described by Selman et al. [32]. In each experiment, ovarian tissue fragments obtained from three females were randomly distributed to experimental groups. All procedures reported here were approved by the Ethics Committee of iBEST. Leibovitz Alectinib molecular weight L-15 was chosen as the base medium for preparing all cryoprotectant solutions tested in our experiment, based on previous studies carried out by Guan et al. [12] and by Seki et al. [30]. To make the medium, Leibovitz L-15 (Sigma) was Gemcitabine cost diluted to 90% and the pH was adjusted to

9.0 using NaOH. Vitrifying tendency of methanol, ethanol, dimethyl sulfoxide (Me2SO), propylene glycol and ethylene glycol solutions made up in L-15 medium was tested at the following range of concentrations (Table 1). Cryo-solutions were tested for vitrification by using three different devices: Plastic straw: 0.25 ml plastic straws (IMV Technologies, L’Aigle, France; reference 005565) were filled at room temperature (22 °C) by suction with a 5 ml syringe. The loaded straws were plunged directly into liquid nitrogen, held for 1 min and then the warming was performed by plunging the straws into a water bath maintained at 28 °C. Vitrification Block™: by using a pipette, a 5 μl droplet was transferred to the hook at the end of a custom designed fibre named Fibreplug™ (CryoLogic Ltd, Melbourne, Australia). The vitrification block was chilled to liquid nitrogen temperature and the fibreplug holding a microdrop was placed on the chilled surface directly, where it was held for 1 min.